Polynucleotide sequence assay

ABSTRACT

Disclosed are methods for detecting or quantifying one or more target polynucleotide sequences in a sample. In one aspect, a sample is contacted with first and second probe pair that are capable of hybridizing to a selected target sequence and a corresponding complementary sequence, respectively. Probe cleavage and ligation results in the formation of ligation products which can be generated in an exponential fashion when the target sequence and/or complement are present in the sample. In another embodiment, a single probe pair can be used to form ligation product in a linear fashion from a complementary template. Reagents and kits are also disclosed.

[0001] This application claims the benefit of priority of U.S.provisional application Serial No. 60/216,514 filed Jul. 3, 2000, whichis incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to methods for detecting orquantifying one or more polynucleotide sequences in one or more samples,and to reagents and kits for use therein.

REFERENCES

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INTRODUCTION

[0055] Methods for detection and analysis of target nucleic acids havefound wide utility in basic research, clinical diagnostics, forensics,and other areas. One important use is in the area of geneticpolymorphism. Genetic polymorphisms generally concern the geneticsequence variations that exist among homologous loci from differentmembers of a species. Genetic polymorphisms can arise through themutation of genetic loci by a variety of processes, such as errors inDNA replication or repair, genetic recombination, spontaneous mutations,transpositions, etc. Such mutations can result in single or multiplebase substitutions, deletions, or insertions, as well as transpositions,duplications, etc.

[0056] Single base substitutions (transitions and transversions) withingene sequences can cause missense mutations and nonsense mutations. Inmissense mutations, an amino acid residue is replaced by a differentamino acid residue, whereas in nonsense mutations, stop codons arecreated that lead to truncated polypeptide products. Mutations thatoccur within signal sequences, e.g., for directing exon/intron splicingof mRNAs, can produce defective splice variants with dramaticallyaltered protein sequences. Deletions, insertions, and other mutationscan also cause frameshifts in which contiguous residues encodeddownstream of the mutation are replaced with entirely different aminoacid residues. Mutations outside of exons can interfere with geneexpression and other processes.

[0057] Genetic mutations underlie many disease states and disorders.Some diseases have been traced directly to single point mutations ingenomic sequences (e.g., the A to T mutation associated with sickle cellanemia), while others have been correlated with large numbers ofdifferent possible polymorphisms located in the same or differentgenetic loci (e.g., cystic fibrosis). Mutations within the same geneticlocus can produce different diseases (e.g., hemoglobinopathies). Inother cases, the presence of a mutation may indicate susceptibility toparticular condition for a disease but is insufficient to reliablypredict the occurrence of the disease with certainty. Most knownmutations have been localized to gene-coding sequences, splice signals,and regulatory sequences. However, it is expected that mutations inother types of sequences can also lead to deleterious, or sometimesbeneficial, effects.

[0058] The large number of potential genetic polymorphisms poses asignificant challenge to the development of methods for identifying andcharacterizing nucleic acid samples and for diagnosing and predictingdisease. In other applications, it is desirable to detect the presenceof pathogens or exogenous nucleic acids and to detect or quantify RNAtranscipt levels.

[0059] In light of the increasing amount of sequence data that isbecoming available for various organisms, and particularly for higherorganisms such as humans, there is a need for rapid and convenientmethods for determining the presence or absence of target mutations.Ideally, such a method should have high sensitivity, accuracy, andreproducibility. Also, the method should allow simultaneous detection ofmultiple target sequences in a single reaction mixture.

SUMMARY OF THE INVENTION

[0060] In one aspect, the invention includes a method for detecting atarget polynucleotide sequence. In the method, a target polynucleotidestrand region and a target-complementary strand region are reacted witha first probe pair and a second probe pair under conditions effectivefor the first probe pair to hybridize to the first and second regions inthe target strand region, forming a first hybridization complex, and forthe second probe pair to hybridize to the first and second regions inthe target-complementary strand region, forming a second hybridizationcomplex. In one embodiment, the first probe pair comprises (i) a firstpolynucleotide probe containing a sequence that is complementary to afirst target region in the target strand region and (ii) a secondpolynucleotide probe comprising a sequence that is complementary to asecond target region in the target strand region, wherein the secondregion is located 5′ to the first region and overlaps the first regionby at least one nucleotide base, and the second probe pair may comprise(i) a third polynucleotide probe containing a sequence that iscomplementary to a first region in the target-complementary strandregion and (ii) a fourth polynucleotide probe containing a sequence thatis complementary to a second region in the target-complementary strandregion, wherein the second region is located 5′ to the first region andoverlaps the first region by at least one nucleotide base.

[0061] Following hybridization, the second probe in the firsthybridization complex and the fourth probe in the second hybridizationcomplex can be cleaved to form (i) a third hybridization complexcomprising the target strand region, the first probe, and a firstfragment of the second probe having a 5′ terminal nucleotide locatedimmediately contiguous to a 3′ terminal nucleotide of the first probe,and (ii) a fourth hybridization complex comprising thetarget-complementary strand region, the third probe, and a firstfragment of the fourth probe having a 5′ terminal nucleotide locatedimmediately contiguous to a 3′ terminal nucleotide of the third probe.The first probe may then be ligated to the hybridized fragment of thesecond probe to form a first ligated strand hybridized to the targetstrand region, and the third probe can be ligated to the fragment of thefourth probe to form a second ligated strand hybridized to thetarget-complementary strand region.

[0062] Denaturation of the first ligated strand from the target strandregion, and of the second ligated strand from the target-complementarystrand region, provides single stranded templates that can be hybridizedto unreacted first and second probe pairs for additional probe cleavageand ligation, thereby increasing the amount of ligated probes. Theoccurrence of template-dependent ligation is evidence that the targetsequence (or its complement) is present in a sample.

[0063] In one embodiment, the first region in the target strand regionoverlaps the second region in the target strand region by a singlenucleotide base, and/or the first region in the target-complementarystrand region overlaps the second region in the target-complementarystrand region by a single nucleotide base. In another embodiment, thefirst region in the target strand region overlaps the second region inthe target strand region by two nucleotide bases, and/or the firstregion in the target-complementary strand region overlaps the secondregion in the target-complementary strand region by two nucleotidebases.

[0064] In another embodiment, the 5′ ends of the first and third probesterminate with a group other than a nucleotide 5′ phosphate group, suchas a nucleotide 5′ hydroxyl group. In another embodiment, the 5′ ends ofthe second and fourth probes terminate with a group other than anucleotide 5′ phosphate group. In another embodiment, the 5′ ends of thefirst, second, third and fourth probes terminate with a group other thana nucleotide 5′ phosphate group.

[0065] In another embodiment, the 3′ ends of the second and fourthprobes terminate with a group other than a nucleotide 3′ hydroxyl group,such as a nucleotide 3′ phosphate group. In another embodiment, the 3′ends of the second and fourth probes terminate with a group other than anucleotide 3′ phosphate group. In another embodiment, the 3′ ends of thefirst, second, third and fourth probes terminate with a group other thana nucleotide 3′ phosphate group.

[0066] In one embodiment, the first, second, third and fourth probes areprovided as covalently separate entities. In another embodiment, thefirst probe pair comprises a first probe and a second probe incovalently linked form, such that the first probe is covalently linkedby its 5′ end to the 3′ end of the second probe by a linking moiety.Similarly, the second probe pair may comprise a third probe and a fourthprobe in covalently linked form, such that the third probe is covalentlylinked by its 5′ end to the 3′ end of the fourth probe by a linkingmoiety.

[0067] In another embodiment, at least one of the probes contains adetectable label. For example, at least one of the first probe or thethird probe may contain a detectable label. Similarly, at least one ofthe second probe and the fourth probe may contain a detectable label.Preferably, the label is a non-radioactive label, and more preferably isa fluorescent label, although any suitable label can be used.

[0068] In practicing the present invention, any method may be employedto detect or measure probe ligation. In one embodiment, probe cleavageproduces a second fragment from the second probe which does notassociate with the third hybridization complex. Detection or measurementof the second fragment, directly or indirectly, is an indication thatthe target sequence is present. An increased amount of the secondfragment in a reaction mixture can also be used to measure the extent ofprobe ligation in prior cycles of cleavage and ligation. Similarly,cleavage of the fourth probe can produce a fourth fragment which doesnot associate with the fourth hybridization complex. Detection ormeasurement of the fourth fragment, or of both the second and fourthfragments, directly or indirectly, can indicate that the target sequenceis present.

[0069] In one embodiment, at least one of the second probe and thefourth probe contains both (i) a fluorescent dye and (ii) a quencher dyewhich is capable of quenching fluorescence emission from the fluorescentdye when the fluorescent dye is subjected to fluorescence excitationenergy, and said cleaving severs a covalent linkage between thefluorescent dye and the quencher dye in the second probe and/or fourthprobe, thereby increasing an observable fluorescence signal from thefluorescent dye.

[0070] In one embodiment, the second fragment is immobilized on a solidsupport for detection. In other embodiments, the second fragment isdetected using electrophoresis or mass spectrometry.

[0071] The second fragment may be detected at any suitable time, such ascontinuously, or after a selected period of time, or after a selectednumber of cycles.

[0072] In another embodiment, the first ligated strand, the secondligated strand, or both, are measured after at least one cycle. Forexample, ligated strands can be immobilized on a solid support fordetection, or can be detected using electrophoresis or massspectrometry. In one embodiment, each detected ligated strand contains afluorescent label.

[0073] In yet another embodiment, the reacting step further comprisesproviding a fifth polynucleotide probe which is complementary to asequence variant of a sequence region to which either the first probe,second probe, third probe, or fourth probe is complementary. Forexample, the fifth polynucleotide probe and the first polynucleotideprobe can be complementary to alternative polymorphic sequences. In onepreferred embodiment, the fifth polynucleotide probe and the firstpolynucleotide probe contain different 3′ terminal nucleotides that arecomplementary to alternative target nucleotide bases. Furthermore, thefirst and fifth polynucleotide probes may contain first and seconddetectable labels that are distinguishable from each other.Alternatively, the fifth and second polynucleotide probes can becomplementary to alternative polymorphic sequences.

[0074] The first and second probe sets can be designed so that cleavageproduces cleaved probes having flush abutting ends or staggered abuttingends. For example, to produce flush abutting ends, the first and thirdprobes may be designed so that the 5′ terminal base of the first regionof the target strand region abuts the 5′ terminal base of said firstregion of the target-complementary strand region. Alternatively,staggered ends can be produced when the 5′ terminal base of the firstregion of the target strand region is separated by one or more basesfrom the 5′ terminal base of the first region of thetarget-complementary strand region.

[0075] In yet another embodiment, the first and second probe pairs takentogether constitute a first probe set, and the method further comprisesreacting a sample with a plurality of different probe sets which areeach designed to detect a different target polynucleotide sequence whichmay be present in the sample. In one embodiment, the method includesdetecting at least one ligated strand produced by each different probeset when the corresponding target sequence is present. In oneembodiment, ligated strands from different probe sets are detected bymass spectrometry. In another embodiment, ligated strands from differentprobe sets are detected by electrophoresis, based on distinct labels orelectrophoretic mobilities, for example.

[0076] The first, second, third, or fourth probe in each probe set canbe immobilized on distinct solid support regions. For example, prior tothe reacting step, a probe from each set is immobilized on a distinctsolid support region. In one embodiment, a probe in each probe setcontains a distinct polynucleotide tag that identifies that probe set.These tags can be used to immobilize the probes to distinct solidsupport regions before, during, or preferably after cycles of cleavageand ligation, to facilitate detection of different target sequences.Such tags can be attached via any suitable position in the probes, suchas the 5′ end of the first probe in each different probe set, or the 3′end of the second probe in each different probe set, for example.

[0077] In another embodiment, for each probe set, the cleaving stepreleases a second fragment from the second probe of each probe set, andthe method further includes detecting a second fragment for each targetthat is present. In one embodiment, the second fragments from differentprobe sets are detected by mass spectrometry. In another embodiment,second fragments are detected by electrophoresis, based on distinctlabels, distinct electrophoretic mobilities, or both.

[0078] In another embodiment, a second fragment from each probe setcontains a distinct polynucleotide tag that identifies that probe set.The tags can be used to immobilize the probes to distinct solid supportregions, as above.

[0079] In another aspect, the methods described herein are modified sothat the second probe pair is omitted, and one or more cycles of probehybridization, cleavage, and ligation produce ligated strands at a ratethat is linearly proportional to the number of cycles.

[0080] For example, the invention also includes a method for detecting atarget polynucleotide sequence, comprising (a) reacting a targetpolynucleotide strand region with a first probe pair of the typedescribed above, under conditions effective for the first and secondprobes in the probe pair to hybridize to the first and second regions inthe target strand region, respectively, forming a first hybridizationcomplex, (b) cleaving the second probe in the first hybridizationcomplex to form (i) a second hybridization complex comprising the targetstrand region, the first probe, and a first fragment of the second probehaving a 5′ terminal nucleotide located immediately contiguous to a 3′terminal nucleotide of the first probe, (c) ligating the first probe tothe hybridized fragment of the second probe to form a first ligatedstrand hybridized to the target strand region, (d) denaturing the firstligated strand from the target strand region, and (e) performing one ormore additional cycles of steps (a) through (d), with the proviso thatin the last cycle, step (d) is optionally omitted.

[0081] Kits and various assay components and reagents are alsocontemplated as discussed farther herein. These and other features andadvantages of the invention will become more readily apparent in lightof the detailed description herein.

DETAILED DESCRIPTION

[0082] The present invention provides methods for detecting orquantifying one or more selected target polynucleotide sequences in asample. The invention is highly accurate, permitting detection of targetsequences with high specificity, and highly sensitive, allowingdetection and/or quantitation of small amounts of target sequences. Theinvention is also advantageous for genotyping and detection of geneticpolymorphisms.

[0083] Definitions

[0084] The following terms or phrases are intended to have the meaningsbelow unless indicated otherwise.

[0085] “Nucleoside” refers to a compound containing a base-pairingmoiety (also referred to as a “base”) such as a purine, deazapurine, orpyrimidine nucleoside base, e.g., adenine, guanine, cytosine, uracil,thymine, deazaadenine, deazaguanosine, inosine, or any functionalequivalent thereof, which is attached to a backbone moiety such as asugar ring or any functional equivalent thereof. Nucleosides includenaturally occurring nucleosides which contain a base-pairing moiety (A,C, G, T or U) linked to the 1′-carbon of a pentose ring, 2′-deoxy and2′-hydroxyl forms thereof (e.g., see Komberg, 1992), and also pentoseanalogs and ring-open equivalents thereof (e.g., see Scheit, 1980;Uhlmann et al., 1990).

[0086] The term “nucleotide” as used herein refers to a phosphate esterof a nucleoside, e.g., a triphosphate ester, wherein the most commonsite of esterification is the pentose 5′-hydroxyl group. In certaincases, term “nucleoside” refers both nucleosides and nucleotides, forconvenience. The terms nucleotide and nucleoside as used herein areintended to include synthetic analogs having modified nucleoside basemoieties, modified sugar moieties, and/or modified phosphate groups andphosphate ester moieties, e.g., as described elsewhere (Scheit 1980;Eckstein, 1991).

[0087] “Polynucleotide” and “oligonucleotide” are interchangeable forpurposes of this text and refer to a polymer of nucleoside monomers,including single, double and triple stranded deoxyribonucleotides,ribonucleotides, α-anomeric forms thereof, and the like. Usually thenucleoside monomers are linked by phosphodiester linkages, such that“phosphodiester linkage” refers to a phosphate ester bond or analogthereof wherein the phosphorous atom is in the +5 formal oxidation stateand one or more of the oxygen atoms is replaced with a non-oxygenmoiety. Exemplary phosphate analogs include phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phosphoranilidate, phosphoramidate,boronophosphates, and the like, including associated counterions, e.g.,H⁺, NH₄ ⁺, Na⁺, and the like, if such counterions are present.“Polynucleotides” and “oligonucleotide” also include polymers ofnon-nucleotidic monomers, linked by phosphate ester or other linkages,which are capable of forming sequence-specific hybrids with a targetnucleic acid, e.g., peptide nucleic acids (PNAs, e.g., see Knudsen,1996). Chimeric structures containing more than one type of linkageand/or nucleotide subunit are also contemplated. Polynucleotidestypically range in size from a few monomeric units, e.g. 8-40, tohundreds or thousands of monomeric units. Whenever a polynucleotide isrepresented by a sequence of letters, such as “ATGCCTG,” it will beunderstood that the nucleotides are in 5′ to 3′ order from left to rightand that “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G”denotes deoxyguanosine, and “T” denotes thymidine, unless otherwisenoted.

[0088] “Polynucleotide probe” refers to any moiety that can hybridize,via hydrogen binding, to a target nucleic acid sequence with sequencespecificity that is suitable for the purposes of the present invention.

[0089] “Target-specific polynucleotide” refers to a polynucleotidehaving a target-binding segment that is perfectly or substantiallycomplementary to a target sequence, such that the polynucleotide bindsspecifically to an intended target without significant binding tonon-target sequences under sufficiently stringent hybridizationconditions.

[0090] “Label” means any moiety that, when attached to a nucleotide orpolynucleotide, renders such nucleotide or polynucleotide detectableusing known detection methods.

[0091] “Diagnosis” is intended to encompass diagnostic, prognostic, andscreening methods.

[0092] “Ligation-incompetent” refers to an entity that, under particularconditions, is incapable of undergoing template-dependent ligation by aligation enzyme.

[0093] “Ligation-blocked” refers to an entity that is chemicallyincapable of undergoing ligation under any conditions until a blockinggroup is removed.

[0094] Samples

[0095] The target nucleic acids for use with the invention may bederived from any organism or other source, including but not limited toprokaryotes, eukaryotes, plants, animals, and viruses, as well assynthetic nucleic acids, for example. The target nucleic acids mayoriginate from any of a wide variety of sample types, such as cellnuclei (e.g., genomic DNA), whole cells, tissue samples, phage,plasmids, mitrochondria, and the like. The target nucleic acids maycontain DNA, RNA, and/or variants or modifications thereof.

[0096] Many methods are available for the isolation and purification oftarget nucleic acids for use in the present invention. Preferably, thetarget nucleic acids are sufficiently free of proteins and any otherinterfering substances to allow adequate target-specific probeannealing, cleavage, and ligation. Exemplary purification methodsinclude (i) organic extraction followed by ethanol precipitation, e.g.,using a phenol/chloroform organic reagent (Ausubel), preferably with anautomated DNA extractor, e.g., a Model 341 DNA Extractor available fromPE Applied Biosystems (Foster City, Calif.); (ii) solid phase adsorptionmethods (Walsh, 1991; Boom); and (iii) salt-induced DNA precipitationmethods (Miller), such methods being typically referred to as“salting-out” methods. Optimally, each of the above purification methodsis preceded by an enzyme digestion step to help eliminate protein fromthe sample, e.g., digestion with proteinase K, or other proteases.

[0097] To facilitate detection, the target nucleic acid can be amplifiedusing a suitable amplification procedure prior to conducting thehybridization, cleavage, and ligation steps of the present invention.Such amplification may be linear or exponential. In a preferredembodiment, amplification of the target nucleic acid is accomplishedusing the polymerase chain reaction (PCR) (e.g., Mullis et al., 1994).Generally, the PCR consists of an initial denaturation step whichseparates the strands of a double stranded nucleic acid sample, followedby repetition of (i) an annealing step, which allows amplificationprimers to anneal specifically to positions flanking a target sequence;(ii) an extension step which extends the primers in a 5′ to 3′ directionthereby forming an amplicon nucleic acid complementary to the targetsequence, and (iii) a denaturation step which causes the separation ofthe amplicon from the target sequence. Each of the above steps may beconducted at a different temperature, preferably using an automatedthermocycler (PE Applied Biosystems, Foster City, Calif.). If desired,RNA samples can be converted to DNA/RNA heteroduplexes or to duplex cDNAby known methods (e.g., Ausubel; Sambrook).

[0098] Method

[0099] The present invention employs probe pairs that are each designedto hybridize to a complementary target sequence, and which are capableof undergoing specific cleavage and ligation when hybridized to thetarget sequence. The probe pairs are useful, for example, in linear andexponential probe ligation methods described herein. Any of a variety ofdifferent probe constructs and configurations can be used, as will bemore fully understood from the following discussion.

[0100] Typically, each probe pair comprises (i) a first polynucleotideprobe containing a sequence that is complementary to a first targetregion in the target strand region and (ii) a second polynucleotideprobe comprising a sequence that is complementary to a second targetregion in the target strand region. The second region is located 5′ tothe first region and overlaps the first region by at least onenucleotide base. Probes can be prepared by any suitable method,preferably using an automated DNA synthesizer and standard chemistries,e.g., phosphoramidite chemistry (Beaucage; Gait, 1984, 1990).

[0101] In one aspect, the invention includes a method for detecting atarget polynucleotide sequence. In the method, a target polynucleotidestrand region is reacted with a first probe pair under conditionseffective for first and second probes of the probe pair to hybridize tothe first and second regions in the target strand region, respectively,forming a first hybridization complex. Following hybridization, thesecond probe in the first hybridization complex can be cleaved to form ahybridization complex comprising the target strand region, the firstprobe, and a first fragment of the second probe having a 5′ terminalnucleotide located immediately contiguous to a 3′ terminal nucleotide ofthe first probe. The first probe may then be ligated to the hybridizedfragment of the second probe to form a first ligated strand hybridizedto the target strand region. After separation of the first ligatedstrand from the target strand region by denaturation, the target strandregion in single-stranded form can be hybridized to a new probe pair foradditional probe cleavage and ligation, thereby increasing the amount ofligated probe. The occurrence of template-dependent ligation is evidencethat the target sequence is present in a sample.

[0102] In one embodiment, the first region overlaps the second region bya single nucleotide base. In another embodiment, the first regionoverlaps the second region by two nucleotide bases. The invention alsocontemplates embodiments in which the first and second regions overlapby 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.

[0103] An exemplary embodiment is illustrated in Scheme I below, whichshows a single-stranded target strand region (T0) aligned with firstpolynucleotide probe (P1) and second polynucleotide probe (P2) of afirst probe pair. The two probes are shown in a 5′ to 3′ orientation(left to right), whereas the target is shown in a 3′ to 5′ orientation.The target strand region, which is the complement of the human ApoBsequence shown in Example 1, contains a first region (R1) and anadjacent region (R2) that is located 5′ to the first region. “X”indicates bases that flank the two target regions and which are nothybridized to the first and second probes.

[0104] In Scheme Ia, the first region and second region overlap by asingle base. The first probe consists of 28 contiguous bases that arecomplementary to the corresponding bases in region R1. The first probepreferably contains a 3′-hydroxyl group (—OH). The second probe consistsof 24 contiguous bases that are complementary to corresponding bases inregion R2. When both the first probe and the second probe are hybridizedto the target strand region, the 3′ terminal A base of the first probeis aligned with the 5′ terminal A base of second probe, such that both Abases may compete for hybridization to the corresponding T base in thetarget strand region (in bold type and underlined). In other words, thetarget-complementary segments of the first and second probes (whichhybridize to R1 and R2) overlap by one base when they are hybridizedfully to the target sequence.

[0105] Hybridization of the first and second probes to the targetsequence produces a ternary complex can be referred to as a “displacedstrand structure”, wherein the overlapping ends of the hybridized firstand second probes can compete for hybridizing to the same complementarybases in the target strand region. It is possible that the overlappingends may exist in an equilibrium between states wherein (i) the 3′ endof the first probe is hybridized directly to the target strand region,displacing the 5′ end of the second probe, (ii) the 5′ end of the secondprobe is hybridized directly to the target strand region, displacing the3′ end of the first probe, (iii) the overlapping ends form a triplexstructure involving Hoogsteen or reverse Hoogsteen basepairing, or (iv)any other possible equilibrium state(s). This hybridization complex (1)is ligation-incompetent, meaning that the abutting ends are not readilyligatable in the presence of a template-dependent ligase enzyme, due tothe absence of abutting, adjacent termini that are matched tocomplementary target bases, and (2) is a substrate for certain 5′nuclease enzymes (referred to herein as 5′ nucleases or 5′ nucleaseenzymes) which recognize hybrid structures containing first and secondpolynucleotide moieties that have overlapping target-complementary endswhen hybridized to a complementary strand. In the presence of such a 5′nuclease, the second probe in the complex can be cleaved to form acleaved hybridization complex comprising the target strand, the firstprobe, and a first fragment of the second probe having a 5′ terminalnucleotide located immediately contiguous to a 3′ terminal nucleotide ofthe first probe.

[0106] With reference to the complex formed upon hybridization of thefirst and second probes with the target strand from Scheme I, cleavagecauses severance of the 5′ terminal base from the second probe, leavingthe first probe and a fragment of the second probe hybridized to thetarget strand. The resultant complex is illustrated in Scheme Ib, inwhich the first probe and the remaining fragment of the second probe areshown on different lines to emphasize that their abutting ends areimmediately contiguous with each other but are not covalently linked.The 5′-end of the remaining fragment from the second probe contains a5′-phosphate group due to the action of the 5′ nuclease.

[0107] The hybridization complex in Scheme Ib is ligation-competentsince the abutting ends of the first probe and the fragment of thesecond probe have chemical groups (a 3′ hydroxyl and a 5′ phosphate)which are amenable to ligation under appropriate conditions. Forexample, treatment of the cleaved complex with a ligase enzyme iseffective to produce a ligated strand (LS 1) hybridized to the targetstrand, as illustrated in Scheme Ic.

[0108] Following denaturation of the ligated strand from the targetstrand, the target strand can be hybridized to a new probe pair, andprobe cleavage and ligation can be repeated to form another ligatedstrand. Formation of such ligated strands indicates that the targetsequence is present in the sample.

[0109] Schemes Ia to Ic illustrate a process that can be carried out toconvert multiple copies of a probe pair (which is present in excessrelative to the amount of the target strand) into ligated strands at alinear rate that depends on the duration of each cycle of hybridization,cleavage, ligation, and denaturation.

[0110] In a further embodiment, exponential production of ligatedstrands can be achieved using first and second probe pairs which aretargeted to a target strand region and a complement of the target strandregion, respectively. An example of such an embodiment is illustratedbelow with reference to Schemes IIa-IId.

[0111] Scheme IIa shows a partial duplex sequence for an “A allele” ofthe gene for human ligase I (“AHL”, for A allele of human ligase I),aligned with complementary first and second probe pairs. The probes (Ap1and Ap2) of the first probe pair are complementary to first and secondregions (R1 and R2) of the target strand region T1. The first and secondprobes (Ap3 and Ap4) of the second probe pair are complementary to firstand second regions (R3 and R4) in the complementary target strand regionT2.

[0112] The first probe (Ap1) of the first probe pair contains 24nucleotides. The 5′ terminal base of Ap1 is matched with respect to acorresponding T base in the target. The second base from the 5′ end ofAp1 is mismatched with respect to a C base in T1. Bases 3 to 24 in Ap1constitute a sequence of 22 contiguous target-complementary bases withrespect to strand region T1. The second probe (Ap2) contains 25nucleotides, of which the 5′-terminal A base is mismatched with respectto a corresponding G base in strand region T1, and the remaining basesconstitute a sequence of 24 contiguous matching bases with respect toT1. Thus, the target regions (Ri and R2) to which the first probe pairbinds are 22 and 24 nucleotides in length, respectively, and overlap bya single base.

[0113] With reference to the second probe pair, the first probe (Ap3)contains 26 nucleotides, of which the first two bases at the 5′ end aremismatched with respect to two G bases in target strand region T2 (thehuman ligase I encoding strand region, which is complementary to targetstrand region T1), and the remaining bases are complementary tocorresponding bases in T2. The second probe (Ap4) contains 23nucleotides, of which the 5′-terminal base is mismatched with respect toa corresponding A base in T2, and the remaining bases are complementaryto corresponding bases in T2. Thus, the target regions (R3 and R4) thefirst and second regions in strand region T2 are 24 and 22 nucleotidesin length, respectively, and the two regions overlap by a single base.

[0114] It will be appreciated that in many situations, designation ofthe two strands of a duplex target as a “target strand” and “complementof the target strand” will be an arbitrary choice, so that reversal ofthese designations may also be appropriate. For example, a gene-codingstrand can be designated as a “target strand” or as a “complement of atarget strand”, depending on the preference of the user. Alternatively,both strands of a target duplex can be referred to as “target strands”.

[0115] Also, when multiple probe pairs are used to detect a multiplepossible target sequences in a sample, it will be appreciated that thedifferent target sequences may be present in the same target strand(i.e., in the sample chromosome or same restriction fragment) or may bepresent in different strands. For this reason, the phrase “targetpolynucleotide strand region” or “target strand region” is used to referto a target sequence regardless of whether two different targetsequences are present in the same strand.

[0116] When both probes of the first probe pair are hybridized to strandregion T1, the 3′ terminal A base of the first probe is aligned with anoverlapping A base at the 5′ end of the target-complementary region ofthe second probe, such that these two overlapping bases may compete forhybridization to the corresponding target T base (in bold type withunderlining) in strand region T1. Similarly, when both probes of thesecond probe pair are hybridized to strand region T2, the 3′ terminal Tbase of the first probe is aligned with an overlapping T base at the 5′end of the target-complementary region of the second probe, such thatthese two overlapping bases may compete for hybridization to thecorresponding target A base (in bold type with underlining) in strandregion T2.

[0117] Hybridization of each probe pair to a complementary sequence inT1 or T2 produces a hybridization complex that is (1)ligation-incompetent, and (2) a substrate for certain 5′ nucleases asdiscussed herein. Prior to hybridization, a duplex target should bedenatured to separate the complementary strands of the target, followedby annealing of the separated strands with their complementary probepairs. The presence of an excess of each probe pair favors formation ofprobe-target complexes and helps minimize reformation of the targetduplex. Resultant complexes are illustrated in Scheme IIb.

[0118] In each complex, the first probe and the remaining fragment ofthe second probe have abutting ends that are immediately contiguous witheach other but are not yet covalently linked. The 5′-end of theremaining fragment from the second probe contains a 5′-phosphate groupdue to the action of the 5′ nuclease enzyme. Each hybridization complexin Scheme IIb is ligation-competent since the abutting ends of the firstprobe and the fragment of the second probe have chemical groups (a 3′hydroxyl and a 5′ phosphate) which are amenable to ligation underappropriate conditions. Ligation of the abutting ends in each complexproduces a ligated strand (LS11 or LS12) hybridized to the target strandregion, as illustrated in Scheme IIc.

[0119] Following separation of the ligated strand from the target strandregion, the ligated strand and target strand region can each behybridized to another first or second probe pair, and the steps of probecleavage and ligation can be repeated to form another ligated strand.After each cycle of hybridization, cleavage, ligation, and strandseparation, the sum of ligated probes is expected to be equal toC×2^(N), where C is the initial amount of the target sequence in thesample, and N is the number of cycles, assuming 100% yield at each step.The formation of ligated strands indicates that the target sequence ispresent in the sample.

[0120] As more cycles of hybridization, cleavage, ligation, and strandseparation are performed, probe ligation products become the predominanttemplate for probe hybridization, cleavage, and ligation, and theproduct mixture becomes dominated by duplexes formed from ligation ofthe first and second probe pairs to form complementary ligated strandsas shown in Scheme IId. Double underlining indicates the A and T basesderived from the 3′-terminal bases of the first probes in the first andsecond probe pairs, respectively.

[0121] It will also be appreciated that if a sample initially containsonly a single-stranded target (i.e., there is no complementary strand),or if the ratio of target to its complementary strand is less than 1:1,then the first cycle of probe hybridization, cleavage, and ligation iseffective to produce a ligated strand that contains a contiguoussequence complementary to the target sequence (target strand region) inthe initial target strand. This ligated, complementary strand can thenserve as a template for binding of a second probe pair to form a ligatedstrand that contains a sequence identical to a corresponding sequence inthe initial target strand.

[0122] As noted above, the first and second probes in each probe paircontain sequences that are complementary to first and second targetregions in the target strand, respectively, such that the target regionsoverlap each other by at least one nucleotide base. The target regionsare preferably selected to be within an invariant target sequence thatwill be present in the sample if the target (e.g., a gene, aheterologous target nucleic acid, or a pathogen nucleic acid) is presentin the sample. Thus, the target-complementary sequence in each probe isusually designed to be perfectly complementary to its respective targetregion. Furthermore, it is preferred that the portions of the probesthat bind the first and second target regions in the vicinity of thecleavage/ligation site be perfectly complementary to the target strandregion. In other words, hybridization may be successful if atarget-complementary sequence is substantially complementary, providedthat site-specific probe cleavage and subsequent ligation are notsignificantly impeded by mismatched bases. However, it may be desirableto design a first or second probe in a probe pair that includes anucleotide base that is deliberately mismatched with respect to thetarget, but is located near (e.g., within 2 to 4 bases of) the locus ofprobe cleavage and ligation (or near a known polymorphic site), todestabilize probe hybridization with non-target sequences.

[0123] By way of illustration only, if the 3′ base of the first probe ismismatched with respect to a corresponding base in the target strandregion, cleavage of a hybridized second probe is possible if the alignedbase in the second probe is within a probe sequence that iscomplementary to the target. However, the presence of the 3′ mismatchcan thereafter inhibit or prevent ligation of the first probe to theremaining fragment of the second probe. If the second region of a targetstrand region contains a base mutation immediately 5′ to the target baseto which the 3′-end of the first probe is complementary, then themismatch with the second probe can prevent stable formation of ahybridization complex that is necessary for site-specific cleavage ofthe second probe.

[0124] In some situations, a target sequence may be susceptible to rapidsequence mutation, as in the case of HIV and other pathogenic organisms.For such situations, the probes should be targeted to a conserved targetregion if possible, or at least to a target region that has a minimumnumber of potential base variations. Alternatively, when potentialsequence variants are known, several different probes can be included inthe reaction, each targeted to a different target sequence variant, toensure that the target sequence is detected. Similar strategies can beused to detect allelic variants and single nucleotide polymorphisms(SNPs) as appropriate.

[0125] It should also be noted that when first and second probe pairsare used that are complementary to the two complementary strands of atarget duplex (as in Schemes IIa to IId), the two probe pairs can bedesigned such that the cleavage sites in the second probes of each pairare directly aligned with each other, or are staggered relative to eachother. For example, the 3′ ends of the first probes in the first andsecond probe pairs in Scheme IIa (Ap1 and Ap3) overlap each other by asingle base. Overlaps of more than one base (e.g., 2, 3, 4, or morebases) are also contemplated. Similarly, probe pairs can also bedesigned such that the 3′ ends of the second probes in each pair abuteach other (zero overlap), or are recessed relative to each other tocreate gaps of one or more bases between their 3′ ends (e.g., gaps of 1,2, 3, 4, 5 or more bases). The choice of probe design to achieve aparticular overlap or recess can be optimized for particularexperimental conditions and sample to reduce no-template or non-specificligations by adjusting temperature, cycle times, and other experimentalparameters as desired.

[0126] The length of the target-complementary sequence in each probe isselected to ensure specific hybridization of the probe to the desiredtarget region, preferably without significant cross-hybridization tonon-target sequences in the sample. One advantage of using atarget-specific probe pair to detect a target sequence is that bothprobes must bind to first and second regions of a target sequence. Ifonly one of the first and second probes is hybridized to a complementarystrand region, then cleavage and ligation to the other probe does notoccur.

[0127] The target complementary sequences of the probes can be of anylength suitable for practice of the invention. In general, the lengthsof the target-complementary sequences in the probes should besufficiently long to allow specific detection of the target sequence,without significant interference from hybridization to non-targetsequences. Typically, the target-complementary sequence in a probe is atleast 8, 10, 15, or 18 nucleotides in length. Preferred length rangesfor the target-complementary sequences are 8 to 40 nucleotides, 10 to 35nucleotides, 15 to 30 nucleotides, and 18 to 24 nucleotides. When twoprobe pairs are used to bind to the two strands of a target duplex,respectively, or when multiple probe pairs are used to detect differenttarget sequences, the melting temperatures of the probes, whenhybridized to their complementary target sequences, preferably fallwithin a ΔTm range (Tmax-Tmin) of 15° C. or less, 10° C. or less, andpreferably 5° C. or less. Probe pairs that have similar meltingtemperatures are also advantageous to obtain better uniformity inhybridization kinetics, so that within-cycle yields are comparable fordifferent probe pairs.

[0128] Melting temperatures of probes can be calculated using knownmethods for predicting oligonucleotide melting temperatures (Breslauer,1986; Rychlik, 1989 and 1990; Wetmur, 1991; Osborne, 1991; Montpetit,1992) for example. Target-complementary probe sequences between about 18and 24 bases in length are preferred because such polynucleotides tendto be very sequence-specific when the annealing temperature is setwithin a few degrees of an oligonucleotide melting temperature(Dieffenbach, 1995). Probe characteristics can be farther optimized byempirical methods, if desired.

[0129] When a hybridization complex has formed between a probe pair anda complementary target sequence, cleavage of the second probe in atarget hybridization complex may be accomplished using any conditionsand reagents that are effective to achieve the desired result.Preferably, cleavage is accomplished using an enzyme from the FEN (5′flap endonuclease) family of enzymes (also referred to as 5′ nucleases,5′ endonucleases, and 5′ exo/endonucleases), or a multi-enzymepolypeptide having FEN activity. For the following discussion,polypeptides that contain FEN activity are referred to collectively as5′ nucleases. Non-polymerase 5′ nucleases can be obtained from E. coli,yeast, mouse, human, Pyrococcus fariosus, Pyrococcus woesei,Methanococcus jannaschii, and Archaeglobus fulgidus (e.g., see D. J.Hosfield et al., J. Biol. Chem. 273:27154 (1998); B. Shen et al., TrendsBiochem. Sci. 23:171 (1998); PCT Pub. WO 98/42873; and U.S. Pat. No.5,874,283 (Harrington et al.)). Numerous DNA polymerase enzymes havebeen shown to contain 5′ nuclease activity, including DNA polymerasesfrom Thermus aquaticus, Thermus flavus, Thermus thermophilus, andBacillus stearothermophilus (e.g., see WO 97/27214, WO 98/23774, and WO98/42873). In many cases, genes for these enzymes have been introducedinto host organisms suitable for expressing large amounts of enzyme.Also useful are truncated or modified DNA polymerase polypeptides whichcan be generated by recombinant or proteolytic methods, and have (i)reduced polymerase activity (but retain nuclease activity) and/or (ii)enhanced 5′ nuclease activity, chimeric and fusion polypeptides with 5′nuclease activity, and 5′ nuclease mutants (e.g., see WO 98/42873). Avariety of enzymes having 5′ nuclease activity are available from ThirdWave Technologies, Madison, Wis., as well as various academiclaboratories.

[0130] The cleavage and ligation steps described herein can be performedunder any appropriate conditions that provide desired results. Bufferconditions can be found in references such as described above withrespect to nuclease cleavage, and in references described below withrespect to ligation (see also Examples 1 and 2 below). Typical buffersinclude Tris, MOPS, Tricine, Bicine, MOBS, and other available buffers(e.g., see Sigma-Aldrich Catalog regarding “Good buffers”). Bufferconcentrations of 5 to 100 mM are typically useful, although higher orlower concentrations can also be used. Salts and other additives, suchas NaCl, LiCl, KCl, glycerol (e.g., 1-10 volume percent) and the likecan also be included if desired, as well as appropriate cofactors forthe particular enzymes that are used (e.g., MgCl₂ or MnCl₂ for somenucleases).

[0131] As noted above, cleavage occurs in the second probe at theinternucleotide linkage located immediately 3′ of the base that alignswith the 3′ most base of the first probe, when the first and secondprobes are hybridized to the correct target sequence. The cleavagereaction produces a fragment of the second probe that remains hybridizedto the target strand region, and also a second fragment of the secondprobe that diffuses from the target strand. If the first and secondtarget regions in the target strand overlap by a single base, then thediffusive second fragment contains the target-complementary base fromthe second probe that was immediately 5′ of the cleavage site, plus anyother groups linked to that base. If the first and second target regionsin the target strand overlap by four target-complementary bases, forexample, then the diffusive second fragment contains, at its 3′ end, thesegment of four target-complementary bases from the second probe thatwere immediately 5′ to the cleavage site, plus any other attachedgroups.

[0132] The cleavage reaction catalyzed by the 5′ nuclease enzyme can bedescribed as being both structure-specific and sequence-specific. Thereaction is structure-specific because the 5′ nuclease enzymespecifically cleaves an internucleotide linkage in the second probebetween a first base that is aligned with the 3′ terminal base of thefirst probe and a second base that is 3′ to the first base, regardlessof the particular sequences of the first probe, second probe, and targetstrand region. The reaction is sequence-specific because the site ofcleavage, relative to the target sequence, is determined by thetarget-complementary sequences of the first and second probes, and alsoby the length of overlap between the target-complementary sequences ofthe probes.

[0133] Preferably, the 5′ nuclease enzyme is a thermostable enzyme thatretains substantially full activity after multiple cycles (e.g., 30cycles) of heating and cooling, so that there is no need to replenish 5′nuclease enzyme during cycling. Such enzymes can be readily obtainedfrom thermophilic organisms as indicated above. In an exemplarypreferred embodiment, the enzyme retains at least 80% of initial 5′nuclease activity after thirty cycles of 65° C. for 1 min.(annealing/cleaving/ligation) and 95° C. for 15 seconds (stranddenaturation).

[0134] According to one embodiment, the second probe in a probe paircontains one or more cleavage-resistant internucleotide linkages toreduce or prevent cleavage of the probes at linkage sites other than theintended cleavage site. Such cleavage-resistant linkages may includephosphorothioates (5′ S, 3′ S, or exo S), phosphorodithioates (e.g.,di-exo or di-endo), phosphoramidates (5′ to 3′ N, 3′ to 5′ N, or exo-N),O-methyl phosphonates, and peptide nucleic acid linkages, etc., forexample. Methods for synthesizing such linkages are well known, and aredescribed, for example, in U.S. Pat. No. 5,837,835 (Gryaznov et al.),U.S. Pat. No. 5,859,233 (Hirschbein et al.), Hunziker et al. (1995), andUhlmann et al. (1990). In one embodiment, the intersubunit linkages ofthe target-complementary portion of the second probe are allcleavage-resistant linkages except for the linkage that is to becleaved. However, the occurrence and extent of probe cleavage atsecondary sites (other than the intended linkage) may besequence-dependent or, for other reasons, may be limited to only a fewlinkages within a probe. These secondary sites can be identified andcharacterized by electrophoretic or other methods, upon whichparticularly susceptible linkages can be replaced withcleavage-resistant linkages, while stable linkages remain as standardphosphodiester linkages.

[0135] It will be appreciated that the effectiveness of a particulartype of linkage may depend on the particular 5′ nuclease that is used.For example, some endonucleases can more readily cleave exo-Sphosphorothioate linkages having Rp chirality than linkages having Spchirality (see Taylor et al., 1985). Also, the presence of one or morePNA linkages close to the target cleavage site may inhibit enzymebinding, so that such linkages may be most useful if located at leastseveral (e.g., at least 5) linkages from the linkage that is to becleaved. The use and placement of a particular linkage type within aprobe is a matter of design choice of the user and the requirements of aparticular assay.

[0136] After site-specific cleavage of the second probe, the first probeand remaining fragment of the second probe are ligated to form a ligatedstrand. The ligation step is accomplished using any suitable conditionsthat are effective to promote covalent ligation of abuttingtarget-complementary termini of contiguously hybridized first probe andcleaved second probe. Usually, ligation can be accomplished enzymicallyusing a ligase enzyme. In a preferred mode, ligation entails couplingthe 3′ terminal 3′ hydroxyl group of the first probe to a 5′ phosphategroup of cleaved second probe, to produce a ligated strand that isconnected by a standard phosphodiester linkage. However, any othercombination of reactive groups can be used, as long as ligation occurswhen the probes are bound to the intended target sequence.

[0137] Numerous ligase enzymes are known in the art and can be obtainedfrom a variety of biological and commercial sources. Exemplary ligasesinclude, but are not limited to, E. coli ligase, T4 ligase, T. aquaticusligase, T. Thermophilus ligase, Pfu ligase, etc. (see, for example, U.S.Pat. No. 5,830,711 (Barany et al.) and EP Patent 320308 B1 (Backman andWang). A thermostable ligase is preferred so that there is no need toreplenish ligase activity during temperature cycling. In an exemplarypreferred embodiment, the enzyme retains at least 80% of initial 5′nuclease activity after thirty cycles of 65° C. for 1 min.(annealing/extension) and 95° C. for 15 seconds (strand denaturation).

[0138] Although it is preferred that ligation is carried out using aligase enyzme, chemical (non-enzymic) ligation is also contemplated. Inone embodiment, chemical ligation can be performed by generating achemically reactive group at the 5′ end of the remaining fragment of thesecond probe that is capable of reacting with a corresponding reactivegroup at the 3′ end of hybridized first probe. When the 3′ base of thefirst probe is immediately contiguous with the 5′ base of the fragmentof the second probe, and the 3′ base and 5′ base are hybridized tomatching bases in the target strand region, the two reactive groups forma covalent linkage due to mutual close proximity. The reaction occurs atthe temperature of the reaction mixture and does not requireillumination with high energy light, although microwave irradiation canbe used to facilitate ligation. For example, the first probe can bedesigned to contain a 3′ bromoacetyl amino group, and the second probecan be designed to contain a phosphorothioate linkage at the site thatis to be cleaved. Cleavage of the second probe produces a 5′thiophosphate group that is immediately contiguous with the 3′bromoacetyl amino group. Displacement of the bromine by the sulfur atomof the thiophosphate group produces a thiophosphorylacylamino linkagebetween the first probe and the remaining second fragment of the secondprobe. The resultant ligated strand can then be detected or can serve asa template for hybridization, cleavage, and ligation of a complementarysecond probe pair. Guidance for exemplary chemical groups that may beused for thermal chemical ligation can be found, for example, in U.S.Pat. No. 5,476,930 (Letsinger and Gryaznov), U.S. Pat. No. 5,741,643(Gryaznov et al.), and references cited therein. Chemical ligation byphotoexcitation is also contemplated, as described in EP Patent 324616B1 (Royer et al.), for example.

[0139] The probe pairs used in the present invention are designed to beligation-incompetent when first and second probes are hybridized totheir corresponding first and second target regions, due to theinability of the two probes to form a nicked duplex structure withsuitably reactive abutting probe ends. A correctly hybridized probe pairbecomes ligation competent only after site-specific cleavage of thesecond probe as discussed above. Generally, the “nicked duplex” that isproduced after cleavage is readily ligated by ligase enzyme (enzymicembodiments) or chemical coupling (chemical embodiments) because theabutting ends of the first probe and cleaved second probe are close toeach other due to hybridization to the target strand region. However,incorrect ligation reactions are also possible due to erroneous probecleavage events, template-independent ligation reactions, andtemplate-dependent reactions resulting from spurious duplex formation.Such side reactions can be inhibited by providing probes with ligationblocked ends. For example, since the 5′ ends of the probes in the probepairs do not participate in target-specific ligation, they can berendered “ligation blocked” by providing 5′ end groups that areincapable of ligation under the reaction conditions of the invention.Thus, in an enzymic ligation embodiment, the probes can be renderedligation blocked by providing a 5′ terminal group that is not anucleotide 5′ phosphate. Such non-ligatable blocking groups can be anyof a large variety of chemical entities, such as 5′ deoxy, 5′ hydroxy,5′ N-acetyl, 5′ O-trityl, 5′ O-monomethoxytrityl, etc. For example, whenfirst and second probe pairs are used that are complementary to bothstrands of a duplex target, the 5′ ends of the first and third probesterminate with a group other than a nucleotide 5′ phosphate group,and/or the 5′ ends of the second and fourth probes terminate with agroup other than a nucleotide 5′ phosphate group, such as 5′ hydroxyl.

[0140] Similarly, the 3′ ends of the second probe in each probe pair canbe rendered ligation-blocked by providing a 3′ terminal group that isnot a nucleotide 3′ hydroxyl group. Exemplary 3′ blocking groups include3′ deoxy, 3′ phosphate, 3′ N-acetyl, 3′ O-trityl, 3′O-monomethoxytrityl, etc.

[0141] In another embodiment, the first and second probes of a probepair are provided in a covalently linked form, such that the first probeis covalently linked by its 5′ end to the 3′ end of the second probe bya linking moiety. In one embodiment, the linking moiety comprises achain of polynucleotides that are not significantly complementary to thetarget strand, the probes, or to any other nucleic acid in the sample.The linking moiety is sufficiently long to allow thetarget-complementary sequences in the probes to hybridize to the targetstrand region and to form a viable hybridization complex for cleavage.Typically, the linking moiety is longer than, preferably at least 10nucleotides longer than, the collective length of the first and secondtarget regions. A polynucleotide linking moiety can contain or consistof any suitable sequence. For example, the linking moiety can be ahomopolymer of C, T, G or A. Alternatively, the linking moiety cancontain or consist of a non-nucleotidic polymer, such as polyethyleneglycol, a polypeptide such as polyglycine, etc.

[0142] In practicing the present invention, the target polynucleotide(s)are preferably converted into single-stranded form by denaturationaccording to known methods, to increase the accessibility of targetsequences for hybridization with the complementary probes. Typically,adequate denaturation is accomplished by heating the sample to anelevated temperature, e.g., at least 90° C. or at least 95° C., for asuitable time, usually at least several seconds to several minutes orlonger if necessary, to sufficiently remove inter- and intra-strandsecondary structure that might otherwise interfere with probehybridization.

[0143] The target strand regions can then be allowed to anneal to thecomplementary probes under conditions effective for the first and secondprobes to hybridize to the first and second regions in the target strandregion, respectively, forming a first hybridization complex, and forthird and fourth probes of a second probe pair (if present) to hybridizeto the first and second regions in the target-complementary strand,respectively, forming a second hybridization complex.

[0144] Hybridization (probe annealing) is performed under conditionswhich are sufficiently stringent to promote sequence specificity, yetsufficiently permissive to allow formation of stable hybrids at anacceptable rate. The temperature and length of time required for probeannealing depend upon several factors including base composition, lengthand concentration of the primer, and the nature of the solvent used,e.g., the concentration of cosolvents such as DMSO (dimethylsulfoxide),formamide, glycerol, and counterions such as magnesium. For example,hybridization (annealing) can be carried out at a temperature that isapproximately 5 to 10° C. below the melting temperature of theprobe-target hybrids in the reaction mixture, although temperaturesoutside this range are also contemplated. For example, even if thereaction temperature is at or above the melting temperatures of firstand second probes, the probes can still transiently form a duplex withthe target that can be correctly cleaved and ligated. Typically, theannealing temperature is in the range of 55° C. to 75° C.

[0145] Probes and probe pairs are provided at any concentration thatprovides the desired result. Each probe pair is provided in excessrelative to the initial amount of target sequence, and also in excessrelative to the final amount of ligated product that is expected in thereaction. Annealing is usually complete within a few seconds or a fewminutes. In one embodiment, the reaction mixture is maintained at aconstant temperature which is suitable for hybridization, site-specificprobe cleavage, and ligation. However, it is also contemplated that thetemperature for probe hybridization can be different from thetemperature or temperatures used for probe cleavage and ligation, orthat other temperature profiles can be used. For example, after aninitial hybridization time period, the temperature can be raised toexpedite the cleavage reaction or ligation reaction, depending on thecharacteristics of the probes and of any enzymes that are used. After asufficient amount of time, the ligated strands can be denatured byelevated temperature as above, followed by cooling to allow the targetstrand regions and ligated probe products to hybridize to new probepairs for another cycle.

[0146] It will be appreciated that due to the presence of excess probe,the target strand region and the target-complementary strand region willanneal to the corresponding complementary probe pairs rather than toeach other. Also, as a result of the annealing conditions, most or allof the excess probes will hybridize to their probe complements. In otherwords, the first probe can hybridize to the third probe, and the secondprobe can hybridize to the fourth probe. Under ordinary circumstances,such probe-probe duplexes do not interfere with the remaining assaysteps.

[0147] If the target strand region is initially present in the sample insingle-stranded form (i.e., lacking a complementary target strand), thenin the contacting step of the first cycle, a hybridization complex willform between the target strand region and one of the probe pairs (e.g.,the first probe pair), while the other probe pair remains in solution.However, after the first cycle of cleavage and ligation, atarget-complementary strand region (the ligation product of the firstprobe and the cleaved second probe) is available for annealing to thesecond probe pair in the next cycle.

[0148] The target sequence or sequences can be detected or quantified inany appropriate way. Target detection or quantification can be based onthe presence of any species or complex that either is not present in thereaction mixture unless the target is present, or is present in anamount greater than the amount of that species that would otherwise bepresent in the absence of the target sequence. By way of example but notlimitation, such detectable species may include:

[0149] 1) A first fragment from the second probe that remains hybridizedto the target strand region after cleavage of the second probe

[0150] 2) A second fragment from the second probe which does notassociate with the third hybridization complex, and the method furtherincludes detecting said second fragment.

[0151] 3) A first hybridization complex, a second hybridization complex,or both, which are formed by hybridization of first and second probepairs, respectively, to complementary target strand regions (or tocomplementary ligated strands)

[0152] 4) A third hybridization complex, a fourth hybridization complex,or both, which are formed after site-specific cleavage of the secondprobe of each probe pair in the complex mentioned in preceding item 3).

[0153] 5) A first ligated strand, a second ligated strand (complementaryto the first), or both, which result from probe ligation.

[0154] Each species can be detected based on a unique property, such aselectrophoretic mobility, mass, or a particular detectable label (ordetectable signal associated with such label). Methods forelectrophoretic separation of nucleic acids and other species are wellknown, and are described, for example in the works of Ausubel (1993, andlater editions) and Sambrook et al. (1989). The invention alsocontemplates the use of probes that have distinct electrophoreticmobilities due to the presence of polymer segments or other moietiesthat confer distinct mobilities to the detected species in sieving ornon-sieving media, as taught in U.S. Pat. Nos. 5,470,705, 5,514,543, and5,580,732 (Grossman et al.), for example.

[0155] In one embodiment, to facilitate detection, at least one probecontains a label. Any suitable label can be used. Labels may be directlabels which themselves are detectable or indirect labels which aredetectable in combination with other agents. Exemplary direct labelsinclude but are not limited to fluorophores, chromophores, radioisotopes(e.g., ³²P, ³⁵S, ³H), spin-labels, chemiluminescent labels, and thelike. Exemplary indirect labels include enzymes which catalyze asignal-producing event, and ligands such as an antigen or biotin whichcan bind specifically with high affinity to a detectable anti-ligand,such as a labeled antibody or avidin. Many comprehensive reviews ofmethodologies for labeling DNA provide guidance applicable to thepresent invention. Such reviews include Matthews et al. (1988); Haugland(1992), Keller and Manak (1993); Eckstein (1991); Kricka (1992), and thelike. Additional methods for creating labeled nucleotides are describedin Fung et al.; Hobbs et al., Menchen et al., and Bergot et al., andRosenblum et al. (all supra).

[0156] In a preferred embodiment, the second probe in at least one probepair contains both (1) a fluorescent dye and (2) a quencher dye thatquenches at least a portion of the fluorescent dye when both dyes arepresent in the probe. The two dyes are positioned in the second probe sothat they are separated by the linkage that is to be specificallycleaved in the cleavage step. Cleavage of the second probe results in afirst fragment which contains most or all of the target-complementarysequence of the probe (and which remains hybridized to the target strandregion), and a second fragment that diffuses from the hybridizationcomplex. Separation of the fluorescent dye and the quencher dye bydiffusion leads to an increased fluorescent signal from the fluorescentdye. Methods for preparing suitable probes containingfluorescer/quencher pairs can be found in Livak et al. (1995) and U.S.Pat. No. 5,876,930 (Livak et al.), for example. Quenchers are alsoavailable from various commercial sources, such as Epoch Biosciences.

[0157] In another embodiment, a first probe in a probe pair can containa donor moiety, and a second probe can contain an acceptor moiety, sothat upon ligation, the donor moiety and acceptor moiety are broughtinto close proximity so that fluorescence emission of the acceptormoiety is increased.

[0158] In another embodiment, a first probe in a probe pair can containa fluorescer moiety, and a second probe can contain a quencher moiety,so that upon ligation, fluorescence emission from the fluorescer moietydecreases due to quenching.

[0159] One or more probes may also contain a member of a specificbinding pair. “Specific binding pair” refers to a pair of molecules thatspecifically bind to one another to form a binding complex. Examples ofspecific binding pairs include, but are not limited to antibody-antigen(or hapten) pairs, ligand-receptor pairs, enzyme-substrate pairs,biotin-avidin pairs, complementary polynucleotide pairs, and the like.The use of a binding pair can be used to attach various labels to theprobe, as discussed above, or to capture the probe on a solid supportthat is coated with the other member of the binding pair.

[0160] In yet another embodiment, ligation is detected or quantifiedusing an intercalating dye such as ethidium bromide or SYBR GREEN(Molecular Probes) or a minor groove binder such as Hoechst 33258, forexample, which are compounds that exhibit increased fluorescence inproportion to the amount of double-stranded nucleic acid in a sample.

[0161] Multiple Targets, Arrays

[0162] The present invention can be used to detect a plurality ofdifferent target sequences in a single sample or in a plurality ofsamples. In one embodiment, different target sequences are detectedseparately in separate reaction mixtures. In another embodiment, asample can be contacted with a plurality of probe sets which are eachdesigned to detect a different target sequence that may be present inthe sample. The various target sequences can be detected based ondetectable characteristics that are unique for each probe pair, such asmass, electrophoretic mobility, fluorescence signal, or a combinationthereof. Methods of electrophoresis are well known and are described,for example, in Ausubel, Sambrook et al. (1989), and Grossman andColburn (1992). The number of target sequences, and corresponding probepairs, that can be used in a single reaction is a matter of choice bythe user, and will depend in part on the resolvability of the propertiesthat are used to distinguish the various reaction products.

[0163] In one embodiment, one of the first probe and second probe of aprobe pair contains a distinct polynucleotide tag (a tag having adefined polynucleotide sequence) that identifies that probe pair. Thetag can be directly attached to the distal end of thetarget-complementary sequence of a probe, or optionally can be linked tothe probe by an intervening spacer group. In another embodiment, the tagis linked to an internal site within the target-complementary sequenceof the probe. Thus, the tag can be linked to an intersubunit linkinggroup, or to a nucleotide base, within a probe. For example, each tagcan be attached to the 5′ end of the first probe in each different probepair, or to the 3′ end of the second probe in each different probe pair.

[0164] Tagged probes or tagged probe fragments can be separated fromeach other by hybridization to corresponding tag complements which areimmobilized on distinct solid support regions. Preferably, the solidsupport regions are configured as an addressable array. By “addressablearray” is meant that the identity of each probe or probe fragment isknown or can be determined from the position of hybridization of thatprobe or probe fragment on the array. Preferably, the tag complementsare immobilized in discrete regions on a planar surface, such that eachdiscrete region contains only tag complements having a particularsequence, and such that the sequence of the tag complement at eachdifferent discrete region is known. Conveniently, the tag complementsare distributed as a periodic two-dimensional array of discrete tagcomplement regions which can be indexed via X and Y coordinates, or anyequivalent thereof.

[0165] Solid phase supports can be formed using any material that allowsfor the tag segments to hybridize specifically to their complementarytag complements on the support. Exemplary support materials includeglass; quartz; silicon; polycarbonate; metallic materials such as GaAs,copper, or germanium; a polymerized gel, such as crosslinkedpolyacrylamide; or membranes such as nylon, polyvinylidine difluoride(PVDF), or poly-tetrafluoroethylene.

[0166] Immobilization of tag-complements in the array is accomplishedusing any of a variety of suitable methods. In one approach, the tagcomplements are deposited onto a solid phase surface using liquiddispensing methods. For example, deposition can be accomplishedrobotically on a poly-lysine-coated microscope slide, followed bytreatment with succinic anhydride to couple the tag complements to thepolylysine moieties, as described by Schena et al. (1995) and Shalon(1995). For covalent attachment, the tag-complements may include asuitably reactive functionality for covalent attachment to the support.Exemplary linking chemistries are disclosed in Barany et al. (1991), Ponet al. (1988), and Menchen et al. (1994).

[0167] In another approach, tag complements can be synthesized on asupport by photolithographic methods, as described in Fodor et al.(1991, 1995), Pirrung et al. (supra), and Shoemaker (1997).Photoremovable groups are attached to a substrate surface, andlight-impermeable masks are used to control the addition of monomers toselected regions of the substrate surface by activating light-exposedregions. Monomer addition to the growing polymer chains is continuedusing different mask arrangements until the desired, different sequencetag complements are formed at the desired addressable locations. Themasking method of Fodor et al. may also be modified to accommodateblock-polymer synthesis. For example, an array of linker groups (e.g., apolypeptide, or an N-protected aminocaproic acid linked to anaminopropyl group) can be formed on the substrate surface viasimultaneous activation of all immobilization regions to form a “carpet”of linker groups. The tag complements are then individually deposited on(or adsorbed to) the substrate surface as liquid drops at selectedaddressable locations, and are exposed to light or heat as appropriateto couple the binding moieties to the immobilized linker groups,preferably while a sufficient amount of solvent still remains from eachdrop.

[0168] Alternatively, the tag complements may be immobilized on thesupport(s) non-covalently, e.g., using ligand-receptor typeinteractions. For example, the tag complements may contain covalentlyattached biotin groups as linker groups, for binding to avidin orstreptavidin polypeptides which have been attached to a support (e.g.,Barrett, 1996).

[0169] Linker segments may also be included between the tag complementsequence and the support to provide a spacer arm which allows thetag-specific binding region to separate from the support, rendering thebinding region more accessible to the sample. Exemplary linker groupsare described, for example, in Fodor et al. (1995) and Brenner (PCTPublications cited above). Preferably, the tag complement is separatedfrom the support by a chain comprising at least 10 chain atoms.

[0170] The support may include depressions in the support for holdingthe deposited tag complements. Elevated protrusions can also be used,onto which the tag complements are deposited. In yet another approach,tag complements can be formed on beads as described in U.S. Pat. No.5,846,719 (Brenner et al.), for example. Alternatively, tag complementsare attached to an array of individual beads attached to a surface, viamagnetic force if the beads are magnetic (Albretsen, 1990), or with anadhesive.

[0171] In another approach, an array is formed on a substrate, such as aglass plate, which is covered with a rectangular array of pieces ofpolyacrylamide gel (e.g., Khrapko et al., 1991). A different tagcomplements is deposited at a selected site and is bound thereto byreacting a 3′-terminal dialdehyde on the tag complements with hydrazidegroups on the polyacrylamide gel piece. Tag complement arrays inaccordance with the invention may also be formed by robotic depositionof tag complements onto nylon (Khrapko et al., supra). Followingdeposition, immobilization of the tag complements may be facilitated byheat or photoactivation as appropriate.

[0172] To reduce the amounts of assay reagents used for tag detection,arrays may be formed as microarrays having tag complement regiondensities of greater than 100 regions/cm², 300 regions/cm², 10³regions/cm², 3×10³ regions/cm², 10⁴ regions/cm², 10⁵ regions/cm², or 10⁶ regions/cm². In addition, the number of different sequence tagcomplements in each array is preferably equal to or greater than 10, 20,50, 100, 200, 500, 1000, 3000, 10,000, 30,000, 100,000, or 300,000.

[0173] The tags and tag complements may be single or double stranded,such that sequence specific hybridization forms either duplexes byWatson and Crick base-pairing, or triplexes by forward or reverseHoogsteen bonding. In embodiments where specific hybridization occursvia triplex formation, coding of tag sequences follows the sameprinciples as for duplex-forming tags. However, there are furtherconstraints on the selection of word sequences. Generally, third strandassociation via Hoogsteen type of binding is most stable alonghomopyrimidine-homopurine tracks in a double stranded target. Usually,base triplets form in T-A*T or C-G*C motifs (where “-” indicatesWatson-Crick pairing and “*” indicates Hoogsteen type of binding);however, other motifs are also possible. For example, Hoogsteen basepairing permits parallel and antiparallel orientations between the thirdstrand (the Hoogsteen strand) and the purine-rich strand of the duplexto which the third strand binds, depending on conditions and thecomposition of the strands. Furthermore, the invention also contemplatesthe use of non-standard base pairing moieties such as disclosed in U.S.Pat. No. 5,432,272 (Benner) and which are available from Erogen as the“AEGIS” system.

[0174] There is extensive guidance in the literature for selectingappropriate sequences, orientation, conditions, nucleoside type (e.g.whether ribose or deoxyribose nucleosides are employed), basemodifications (e.g. methylated cytosine, and the like in order tomaximize, or otherwise regulate, triplex stability as desired inparticular embodiments, e.g., Brenner (supra). More generally,conditions for annealing single-stranded or duplex tags tosingle-stranded or duplex sequence complements are well known, e.g.Brenner (supra), Ji et al. (1993), Cantor et al. (supra), Wetmur (1991),Breslauer et al. (1986), Schena (1995), and the like.

[0175] Detection

[0176] Any detection method may be used which is suitable for the typeof label employed. Thus, exemplary detection methods include radioactivedetection, optical absorbance detection, e.g., UV-visible absorbancedetection, optical emission detection, e.g., fluorescence orchemiluminescence. For example, captured tagged species can be detectedon an array by scanning all or portions of each array simultaneously orserially, depending on the scanning method used. For fluorescencelabeling, selected regions on an array may be serially scannedone-by-one or row-by-row using a fluorescence microscope apparatus, suchas described in Fodor (1995) and Mathies et al. (1992). Hybridizationpatterns may also be scanned using a CCD camera (e.g., ModelTE/CCD512SF, Princeton Instruments, Trenton, N.J.) with suitable optics(Ploem, 1993), such as described in Yershov et al. (1996), or may beimaged by TV monitoring (Khrapko, 1991). For radioactive signals (e.g.,³²P), a phosphorimager device can be used (Johnston et al., 1990;Drmanac et al., 1992; 1993). Other commercial suppliers of imaginginstruments include General Scanning Inc., (Watertown, Mass.,www.genscan.com), Genix Technologies (Waterloo, Ontario, Canada;www.confocal.com), and Applied Precision Inc. Such detection methods areparticularly useful to achieve simultaneous scanning of multiple tagcomplement regions.

[0177] Measured signals can be analyzed manually or by appropriatecomputer methods to tabulate results. The results can be measured toprovide qualitative or quantitative results, depending on the needs ofthe user. Reaction conditions can include appropriate controls forverifying the integrity of hybridization, and for providing standardcurves for quantitation, if desired.

[0178] Kits

[0179] The invention also contemplates kits which are useful inpracticing the invention. Such kits may include one or more probe pairsas discussed above, and optionally, a 5′ nuclease enzyme and a ligaseenzyme. The kit may also include buffers and any other reagents thatfacilitate the method.

[0180] From the foregoing, it can be seen how the features and benefitsof the invention can be achieved. The invention provides a convenientmethod for determining the presence or absence of one or more targetsequences, and for quantification as well. The method is amenable tohigh-throughput processing of many target sequences and many differentsamples. The invention can be used for a variety of purposes, such asgenetic screening, allele determination, sample identification, diseasediagnosis, forensics, agricultural analysis, and many others. The methodcan also be used to establish a sequence profile of one or more samples,for identifying or distinguishing samples.

[0181] The invention is further illustrated by way of certain exampleswhich are not intended to limit the invention in any way.

EXAMPLE 1

[0182] Afu FEN was obtained from Third Wave Technology (Madison, Wis.).AMPLIGASE™ was purchased from Epicentre Technologies. Reaction buffer(1×) contained 10 mM MOPS pH 7.5, 10 μM NAD⁺, 3.0 mM MgCl₂, 0.05% NP-40,0.05% TWEEN-20 (v:v), and 60 nM ROX passive reference (AppliedBiosystems).

[0183] Genomic DNA that was homozygous for the A allele of human ligaseI (from Raji leukemia cells) was obtained from PE Biosystems (DNATemplate Reagent, Part No. 401970). Genomic DNA that was homozygous forthe C allele of human ligase I, or heterozygous for the A and C alleles,was obtained from Coriell Institute for Medical Research.

[0184] Probe sets (see below) were prepared for detecting the followingtarget sequences: Target 1: Human ApoB5′-AAGAAATTATCTCGGTCCTCACAATAAACTGCGAGGTCACTGTGAGTTTTCCT-3′ (SEQ IDNO: 1) Target 2: Human Ligase I (A allele) 5′-AAAGCCTCACAGAGGCTGAAGTGGCA ACAGAGAAGGAAGGAGAAGACGGGG-3′ (SEQ ID NO: 2) Target 3: Human Ligase I(C allele) 5′-AAAGCCTCACAGAGGCTGAAGTGGC C ACAGAGAAGGAAGGAGAAGACGGGG-3′(SEQ ID NO: 3)

[0185] The following probe sets were synthesized by the phosphoramiditesynthesis method, where F=6-Fam, and Q=Tamra, and all probe sequencesare 5′ to 3′ (see Mullah et al., Nucl. Acids Res. 26:1026-1031 (1998);and “Chemical methods for 5′ non-isotopic labelling of PCR probes andprimers” (Chapter 3) by Alex Andrus, in PCR 2: A Practical Approach, M.J. McPherson et al., eds, IRL Press, NY (1995)): ApoB Probe Set(control):      ApoB probe 1: AAGAAATTATCTCGGTCCTCACAATAAAC (SEQ ID NO:4)      ApoB probe 2: F-ACTGCGAGGTCACTGTGAGTTTTC-Q (SEQ ID NO: 5)     ApoB probe 3: AAGAAAACTCACAGTGACCTCGCAG (SEQ ID NO: 6)      ApoBprobe 4: F-AGTTTATTGTGAGGACCGAGATAATTTC-Q (SEQ ID NO: 7) A-Allele ofHuman Ligase (AHL) Probe Set:       AHL probe 1:AACCTCACAGAGGCTGAAGTGGCA (SEQ ID NO: 8)       AHL probe 2:F-AAACAGAGAAGGAAGGAGAAGACGG-Q (SEQ ID NO: 9)       AHL probe 3:AACCGTCTTCTCCTTCCTTCTCTGTT (SEQ ID NO: 10)       AHL probe 4:F-ATGCCACTTCAGCCTCTGTGAGG-Q (SEQ ID NO: 11) C-allele of human ligase(CHL) probe set:        CHL probe 1: CCCCTCACAGAGGCTGAAGTGGCC (SEQ IDNO: 12)        CHL probe 2: F-ACACAGAGAAGGAAGGAGAAGACGG-Q (SEQ ID NO:13)        CHL probe 3: CCCCGTCT TCTCCTTCCTTCTCTGTG (SEQ ID NO: 14)       CHL probe 4: F-AGGCCACTTCAGCCTCTGTGAGG-Q (SEQ ID NO: 15)

[0186] Alignments of the probes with corresponding target sequences areshown below. Only one target strand region is shown for each targetsequence, for which probes 3 and 4 from each probe set arecomplementary. Probes 1 and 2 from each probe set are complementary tothe complement (not shown) of the target strand region. The A base and Cbase that distinguish the A and C alleles, respectively, of the humanligase I gene are highlighted by bold and underlining. Doubleunderlining indicates bases at the 3′ termini of the first and thirdprobes that are invasive with respect to same-sequence bases at or nearthe 5′ terminal ends of the second and fourth probes, respectively.Lower case letters indicate bases which are non-complementary withrespect to the target sequence (or the complement of the target). ApoBAlignment ApoP1: 5′-AAGAAATTATCTCGGTCCTCACAATAAAC-3′ ApoP2                          5′-F-ACTGCGAGGTCACTGTGAGTTTTC-Q-3′ ApoTgt:  AAGAAATTATCTCGGTCCTCACAATAAACTGCGAGGTCACTGTGAGTTTTCCT-3′ ApoP3:                           3′-GACGCTCCAGTGACACTCAAAAGaA′ ApoP4:5′-Q-CTTTAATAGAGCCAGGAGTGTTATTTGA-F-5′ AHL Alignment AHLp1: 5′-AaCCTCACAGAGGCTGAAGTGGCA-3′ AHLp2:                     5′-F-aAACAGAGAAGGAAGGAGAAGACGG-Q-3′ AHLTgt: AAAGCCTCACAGAGGCTGAAGTGGC A ACAGAGAAGGAAGGAGAAGACGGGG-3′ AHLp3:                       3′-TTGTCTCTTCCTTCCTCTTCTGCCaa-5′ AHLp4: 3′-Q-GGAGTGTCTCCGACTTCACCGTa-F-5′ CHL Alignment CHLp1: 5′-cCCCTCACAGAGGCTGAAGTGGCC-3′ CHLp2:                     5′-F-aCACAGAGAAGGAAGGAGAAGACGG-Q-3′ CHLTgt: AAAGCCTCACAGAGGCTGAAGTGGC C ACAGAGAAGGAAGGAGAAGACGGGG-3′ CHLp3:                        3′-GTGTCTCTTCCTTCCTCTTCTGCCCC-5′ CHLp4: 3′-Q-GGAGTGTCTCCGACTTCACCGGa-F-5′

[0187] Reaction mixtures (30 μL each) were prepared containingapproximately 1 to 3 ng of genomic DNA, 100 nM of each probe from theApoB, AHL, or CHL probe set, 1 ng of Afu FEN, and 5 units of AMPLIGASE™in 1× reaction buffer. Reaction tubes were placed in a 96 well opticalplate (Part No. N801-0560) of a PE Biosystems GENEAMP™ System 7700.After heating to 95° C. for 2 min., the reaction mixtures were subjectedto 50 two-step heating cycles of 65° C. for 1 min. and 95° C. for 15sec. For each well, spectra were collected within a wavelength window of500 to 650 nm during each data collection cycle. Each data collectioncycle time lasted about 7 seconds. The spectra recorded for each wellwere deconvoluted to reflect the signal intensities of each individualdye, and the signal intensity for FAM at the end of each cycle wasplotted as a ratio with respect to the signal intensity of ROX, as afunction of cycle number. Results are shown in Table 1 below, which showcalculated values for the cycle number (Ct) in which specific signal wasdetectable over an arbitrarily selected threshold level, such that alower estimated Ct value indicates a higher starting targetconcentration. TABLE 1 Sample Probe Set in Reaction Mixture ZygosityApoB AHL CHL None 37.2 ± 0.4 35.9 ± 0.2 42.8 ± 1.2 A/A 32.2 ± 0.1 30.5 ±0.2 41.0 ± 0.4 A/C 30.3 ± 0.1 30.2 ± 0.2 31.2 ± 0.1 C/C 32.5 ± 0.2 36.0± 0.4 32.7 ± 0.2

[0188] The results demonstrate that the AHL and CHL probe sets providespecific detection of each target-matched genomic sequence.Specifically, a Ct value of 30.5 was observed for the homozygous Asample in the presence of the AHL probe set, whereas the CHL probe setshowed no detectable amplification through 40 cycles. Conversely, a Ctvalue of 32.7 was calculated for the homozygous C sample in the presenceof the CHL probe set, whereas the AHL probe set showed no detectableamplification until about cycle 36. For the heterozygous A/C sample,detectable signals were observed at about the same cycle numbers (about30 or 31) for the AHL and CHL probe sets, respectively, consistent witha 1:1 allelic ratio.

[0189] The late-occurring signals observed with the AHL and CHL probesets in the presence of non-target allelic samples were significantlyshallower in slope than the slopes of the signals produced by thetarget-matched probe sets, and were probably attributable tonon-template background reactions for which signals are provided in therow of data identified as “None”. For example, in the absence of genomicDNA, the AHL probe set produced a Ct value of 35.9, which issubstantially the same as the Ct value of 36.0 obtained for the AHLprobe set reacted with the homozygous C sample. Similarly, in theabsence of genomic DNA, the CHL probe set produced a Ct value of 42.8,which is close to the Ct value of 41.0 obtained for reaction of the CHLprobe set with the homozygous A sample.

[0190] The results for target-matched AHL and CHL probe sets also agreedwell with data obtained with the ApoB probe set, such that the Ct valuesobtained for the ApoB signal were within about 2 cycles of the Ct valuesobserved for target-matched AHL and CHL probe sets. The disparitybetween the ApoB Ct values and the target-matched AHL or CHL values maybe attributable to different amplification efficiencies for differentprobe sets, and pipetting errors. In all cases, the no-sample controlreactions for the different probe sets occurred in much later cyclesthan the Ct cycle numbers, confirming the target-specificity of theprobe sets.

EXAMPLE 2

[0191] The conditions in Example 1 were repeated using differentconcentrations of target DNA homozygous for the C allele of human ligaseI (0, 1, 10, 100, and 1000 copies, each in quadruplicate) in thepresence of the CHL probe set. The results are shown below: TABLE 2Target Copy Number Ct 0 43.7 ± 0.6 1 42.7 ± 0.8 10  39.8 ± 0.1 100  35.6± 0.2 1000   31.2 ± 0.2

[0192] The results show that as expected, the Ct values showed aninverse linear relationship to the log of target copy number, and fewerthan 10 copies could be reliably detected.

[0193] All references cited herein are incorporated by reference as ifeach was separately but expressly incorporated by reference.

[0194] Although the invention has been described with reference toparticular embodiments, it will be appreciated that various changes andmodifications may be made without departing from the scope and spirit ofthe invention.

1. A method for detecting a target polynucleotide sequence, the methodcomprising (a) reacting a target polynucleotide strand region and atarget-complementary strand region with a first probe pair and a secondprobe pair, the first probe pair comprising (i) a first polynucleotideprobe containing a sequence that is complementary to a first targetregion in the target strand region and (ii) a second polynucleotideprobe comprising a sequence that is complementary to a second targetregion in the target strand region, wherein the second region is located5′ to the first region and overlaps the first region by at least onenucleotide base, and the second probe pair comprising (i) a thirdpolynucleotide probe containing a sequence that is complementary to afirst region in the target-complementary strand region and (ii) a fourthpolynucleotide probe containing a sequence that is complementary to asecond region in the target-complementary strand region, wherein thesecond region is located 5′ to the first region and overlaps the firstregion by at least one nucleotide base, under conditions effective forthe first and second probes to hybridize to the first and second regionsin the target strand region, respectively, forming a first hybridizationcomplex, and for the third and fourth probes to hybridize to the firstand second regions in the target-complementary strand region,respectively, forming a second hybridization complex, (b) cleaving thesecond probe in the first hybridization complex, and the fourth probe inthe second hybridization complex, to form (i) a third hybridizationcomplex comprising the target strand region, the first probe, and afirst fragment of the second probe having a 5′ terminal nucleotidelocated immediately contiguous to a 3′ terminal nucleotide of the firstprobe, and (ii) a fourth hybridization complex comprising thetarget-complementary strand region, the third probe, and a firstfragment of the fourth probe having a 5′ terminal nucleotide locatedimmediately contiguous to a 3′ terminal nucleotide of the third probe,(c) ligating the first probe to the hybridized fragment of the secondprobe to form a first ligated strand hybridized to the target strandregion, and ligating the third probe to the fragment of the fourth probeto form a second ligated strand hybridized to the target-complementarystrand region, (d) denaturing the first ligated strand from the targetstrand region and the second ligated strand from thetarget-complementary strand region, and (e) performing one or moreadditional cycles of steps (a) through (d), with the proviso that in thelast cycle, step (d) is optionally omitted.
 2. The method of claim 1,wherein the first region overlaps the second region by one nucleotidebase.
 3. The method of claim 1, wherein the 5′ ends of the first andthird probes terminate with a group other than a nucleotide 5′ phosphategroup.
 4. The method of claim 3, wherein the 5′ ends of the first andthird probes terminate with a nucleotide 5′ hydroxyl group.
 5. Themethod of claim 1, wherein the 5′ ends of the second and fourth probesterminate with a group other than a nucleotide 5′ phosphate group. 6.The method of claim 5, wherein the 5′ ends of the second and fourthprobes terminate with a nucleotide 5′ hydroxyl group.
 7. The method ofclaim 1, wherein the 5′ ends of the first, second, third and fourthprobes terminate with a group other than a nucleotide 5′ phosphategroup.
 8. The method of claim 1, wherein the 3′ ends of the second andfourth probes terminate with a group other than a nucleotide 3′ hydroxylgroup.
 9. The method of claim 8, wherein said 3′ ends of the second andfourth probes terminate with a nucleotide 3′ phosphate group.
 10. Themethod of claim 1, wherein at least one of the probes contains adetectable label.
 11. The method of claim 10, wherein the label is afluorescent label.
 12. The method of claim 10, wherein the label is aradiolabel.
 13. The method of claim 10, wherein the label is achemiluminescent label.
 14. The method of claim 10, wherein the label isan enzyme.
 15. The method of claim 10, wherein at least one of the firstprobe and the third probe contains a detectable label.
 16. The method ofclaim 15, wherein each of the first probe and third probe contains adetectable label.
 17. The method of claim 16, wherein the detectablelabels on the first probe and third probe are the same.
 18. The methodof claim 10, wherein at least one of the second probe and the fourthprobe contains a detectable label.
 19. The method of claim 18, whereineach of the second probe and the fourth probe contains a detectablelabel.
 20. The method of claim 19, wherein the second probe and fourthprobe contain the same detectable label.
 21. The method of claim 1,wherein said cleaving produces a second fragment from the second probewhich does not associate with the third hybridization complex, and themethod further includes detecting said second fragment.
 22. The methodof claim 21, wherein at least one of the second probe and the fourthprobe contains both (i) a fluorescent dye and (ii) a quencher dye whichis capable of quenching fluorescence emission from the fluorescent dyewhen the fluorescent dye is subjected to fluorescence excitation energy,and said cleaving severs a covalent linkage between the fluorescent dyeand the quencher dye in the second probe and/or fourth probe, therebyincreasing an observable fluorescence signal from the fluorescent dye.23. The method of claim 22, wherein the second probe and the fourthprobe each contain (i) a fluorescent dye and (ii) a quencher dye. 24.The method of claim 21, wherein said cleaving further produces a secondfragment from the fourth probe which does not associate with the fourthhybridization complex, and the method further includes detecting bothsecond fragments.
 25. The method of claim 21, wherein said secondfragment comprises one or more contiguous nucleotides which aresubstantially non-complementary to the target strand region.
 26. Themethod of claim 25, wherein said one or more contiguous nucleotidescomprise 1 to 20 nucleotides.
 27. The method of claim 21, which furtherincludes immobilizing the second fragment on a solid support.
 28. Themethod of claim 21, which further includes subjecting the secondfragment to electrophoresis.
 29. The method of claim 21, which furtherincludes detecting the second fragment by mass spectrometry.
 30. Themethod of claim 21, which comprises detecting the second fragment afterthe last cycle.
 31. The method of claim 21, which comprises detectingthe second fragment during or after a plurality of cycles.
 32. Themethod of claim 31, which comprises detecting the second fragment duringall of the cycles.
 33. The method of claim 1, which further includesdetecting the first hybridization complex, the second hybridizationcomplex, or both, after at least one cycle.
 34. The method of claim 1,which further includes detecting the third hybridization complex, thefourth hybridization complex, or both, after at least one cycle.
 35. Themethod of claim 1, which further includes detecting the first ligatedstrand, the second ligated strand, or both, after at least one cycle.36. The method of claim 35, wherein said detecting comprises anelectrophoretic separation step.
 37. The method of claim 35, wherein thefirst ligated strand, the second ligated strand, or both, are detectedby mass spectrometry.
 38. The method of claim 35, wherein the firstligated strand is detected.
 39. The method of claim 38, wherein thefirst ligated strand contains a fluorescent label.
 40. The method ofclaim 35, wherein the first ligated strand and second ligated strand aredetected, and each ligated strand contains a detectable label.
 41. Themethod of claim 40, wherein each detectable label is a fluorescentlabel.
 42. The method of claim 35, wherein the first ligated strand, thesecond ligated strand, or both, are immobilized on a solid support. 43.The method of claim 42, wherein after the last cycle, the first ligatedstrand is immobilized on the solid support and detected.
 44. The methodof claim 42, wherein said reacting comprises providing the first probeor second probe immobilized on the solid support, so that the firstligated strand is immobilized on the solid support.
 45. The method ofclaim 1, wherein the first probe pair comprises a first probe and asecond probe in covalently linked form, such that the first probe iscovalently linked by its 5′ end to the 3′ end of the second probe by alinking moiety.
 46. The method of claim 45, wherein the second probepair comprises a third probe and a fourth probe in covalently linkedform, such that the third probe is covalently linked by its 5′ end tothe 3′ end of the fourth probe by a linking moiety.
 47. The method ofclaim 45, wherein the linking moiety comprises a chain ofpolynucleotides that is not substantially complementary to the targetpolynucleotide.
 48. The method of claim 1, wherein said reacting furthercomprises providing a fifth polynucleotide probe which is complementaryto a sequence variant of a region to which either the first probe,second probe, third probe, or fourth probe is complementary.
 49. Themethod of claim 48, wherein the fifth polynucleotide probe and the firstpolynucleotide probe are complementary to alternative polymorphicsequences in the target polynucleotide strand region.
 50. The method ofclaim 49, wherein the target complementary sequences of the fifthpolynucleotide probe and the first polynucleotide probe containdifferent 3′ terminal nucleotides that are complementary to alternativetarget nucleotide bases in the alternative polymorphic sequences. 51.The method of claim 49, wherein the first polynucleotide probe containsa first detectable label, and the fifth polynucleotide probe contains asecond detectable label that is distinguishable from the firstdetectable label.
 52. The method of claim 51, wherein said labels arefluorescent labels.
 53. The method of claim 48, wherein the fifthpolynucleotide probe and the second polynucleotide probe arecomplementary to alternative polymorphic sequences in the targetpolynucleotide strand region.
 54. The method of claim 53, wherein thetarget complementary sequences of the fifth polynucleotide probe and ofthe second polynucleotide probe contain different 5′ terminalnucleotides that are complementary to alternative target nucleotidebases in the alternative polymorphic sequences.
 55. The method of claim53, wherein the second polynucleotide probe contains a first detectablelabel, and the fifth polynucleotide probe contains a second detectablelabel that is distinguishable from the first detectable label.
 56. Themethod of claim 55, wherein said labels are fluorescent labels.
 57. Themethod of claim 1, wherein the 5′ terminal base of said first region ofthe target strand region abuts the 5′ terminal base of said first regionof the target-complementary strand region.
 58. The method of claim 1,wherein the 5′ terminal base of said first region of the target strandregion is separated from the 5′ terminal base of said first region ofthe target-complementary strand region by at least one nucleotide base.59. The method of claim 1, wherein said reacting further includesproviding a third probe pair which is complementary to a second targetpolynucleotide strand region and a fourth probe pair which iscomplementary to a complement of the second target polynucleotide strandregion, the third probe pair comprising (i) a fifth polynucleotide probecontaining a sequence that is complementary to a first target region inthe second target strand region and (ii) a sixth polynucleotide probecomprising a sequence that is complementary to a second target region inthe second target strand region, wherein the second region is located 5′to the first region and overlaps the first region by at least onenucleotide base, and the fourth probe pair comprising (i) a seventhpolynucleotide probe containing a sequence that is complementary to afirst region in the second said target-complementary strand region and(ii) an eighth polynucleotide probe containing a sequence that iscomplementary to a second region in the second said target-complementarystrand region, wherein the second region is located 5′ to the firstregion and overlaps the first region by at least one nucleotide base,under conditions effective for the fifth and sixth probes to hybridizeto the first and second regions in the second target strand region,respectively, forming a fifth hybridization complex, and for the seventhand eighth probes to hybridize to the first and second regions in thesecond said target-complementary strand region, respectively, forming asixth hybridization complex if the second said target-complementarystrand region is present in the sample.
 60. The method of claim 1,wherein the first probe pair and the second probe pair taken togetherconstitute a first probe set, and the method further comprises reactinga sample with a plurality of different probe sets which are eachdesigned to detect a different target polynucleotide sequence which maybe present in the sample.
 61. The method of claim 60, wherein saiddetecting comprises detecting at least one ligated strand produced byeach different probe set when the corresponding target sequence ispresent.
 62. The method of claim 61, wherein ligated strands fromdifferent probe sets are detected by mass spectrometry.
 63. The methodof claim 61, wherein ligated strands from different probe sets aredetected by electrophoresis.
 64. The method of claim 63, wherein ligatedstrands from different probe sets have different electrophoreticmobilities.
 65. The method of claim 64, wherein ligated strands fromdifferent probe sets contain detectable labels which may be the same ordifferent.
 66. The method of claim 65, wherein the labels arefluorescent labels.
 67. The method of claim 63, wherein ligated strandsfrom at least two different probe sets contain different fluorescentlabels.
 68. The method of claim 60, wherein prior to step (a), the firstprobe, the second probe, the third probe, or the fourth probe from thedifferent probe sets is immobilized on a distinct solid support region.69. The method of claim 60, wherein one of the first probe, the secondprobe, the third probe, or the fourth probe in each probe set contains adistinct polynucleotide tag that identifies that probe set.
 70. Themethod of claim 69, which comprises hybridizing species containing saidtags to a plurality of corresponding tag complements which areimmobilized on distinct solid support regions.
 71. The method of claim70, wherein each distinct polynucleotide tag is attached to the 5′ endof the first probe in each different probe set.
 72. The method of claim70, wherein each distinct polynucleotide tag is attached to the 3′ endof the second probe in each different probe set.
 73. The method of claim70, wherein said distinct solid support regions are located on asubstantially planar surface.
 74. The method of claim 70, wherein saiddistinct solid support regions are located on different beads.
 75. Themethod of claim 60, wherein for each probe set, said cleaving produces asecond fragment from the second probe of the probe set which does notassociate with the third hybridization complex, and the method furtherincludes detecting the second fragment.
 76. The method of claim 75,wherein second fragments from different probe sets are detected by massspectrometry.
 77. The method of claim 75, wherein second fragments fromdifferent probe sets are detected by electrophoresis.
 78. The method ofclaim 77, wherein second fragments from different probe sets havedifferent electrophoretic mobilities.
 79. The method of claim 77,wherein second fragments from different probe sets contain detectablelabels which may be the same or different.
 80. The method of claim 79,wherein the labels are fluorescent labels.
 81. The method of claim 75,wherein second fragments from at least two different probe sets containdifferent fluorescent labels.
 82. The method of claim 75, wherein thesecond fragment from each different probe set contains a distinctpolynucleotide tag that identifies the probe set.
 83. The method ofclaim 82, which comprises hybridizing second fragments containing saidtags to a plurality of corresponding tag complements which areimmobilized on distinct solid support regions.
 84. The method of claim83, wherein said distinct solid support regions are located on asubstantially planar surface.
 85. The method of claim 83, wherein saiddistinct solid support regions are located on different beads.
 86. Amethod for detecting a target polynucleotide sequence, the methodcomprising (a) reacting a target polynucleotide strand region with afirst probe pair, the first probe pair comprising (i) a firstpolynucleotide probe containing a sequence that is complementary to afirst target region in the target strand region and (ii) a secondpolynucleotide probe comprising a sequence that is complementary to asecond target region in the target strand region, wherein the secondregion is located 5′ to the first region and overlaps the first regionby at least one nucleotide base, under conditions effective for thefirst and second probes to hybridize to the first and second regions inthe target strand region, respectively, forming a first hybridizationcomplex, (b) cleaving the second probe in the first hybridizationcomplex to form (i) a second hybridization complex comprising the targetstrand region, the first probe, and a first fragment of the second probehaving a 5′ terminal nucleotide located immediately contiguous to a 3′terminal nucleotide of the first probe, (c) ligating the first probe tothe hybridized fragment of the second probe to form a first ligatedstrand hybridized to the target strand region, (d) denaturing the firstligated strand from the target strand region, and (e) performing one ormore additional cycles of steps (a) through (d), with the proviso thatin the last cycle, step (d) is optionally omitted.
 87. The method ofclaim 86, wherein the first region overlaps the second region by onenucleotide base.
 88. The method of claim 86, wherein the 5′ end of thefirst probe terminates with a group other than a nucleotide 5′ phosphategroup.
 89. The method of claim 88, wherein the 5′ end of the first probeterminates with a nucleotide 5′ hydroxyl group.
 90. The method of claim86, wherein the 5′ end of the second probe terminates with a group otherthan a nucleotide 5′ phosphate group.
 91. The method of claim 90,wherein the 5′ end of the second probe terminates with a nucleotide 5′hydroxyl group.
 92. The method of claim 86, wherein the 5′ end of thefirst and second probes terminate with a group other than a nucleotide5′ phosphate group.
 93. The method of claim 86, wherein the 3′ end ofthe second probe terminates with a group other than a nucleotide 3′hydroxyl group.
 94. The method of claim 93, wherein said 3′ end of thesecond probe terminates with a nucleotide 3′ phosphate group.
 95. Themethod of claim 86, wherein at least one of the probes contains adetectable label.
 96. The method of claim 95, wherein the label is afluorescent label.
 97. The method of claim 95, wherein the label is aradiolabel.
 98. The method of claim 95, wherein the label is achemiluminescent label.
 99. The method of claim 95, wherein the label isan enzyme.
 100. The method of claim 95, wherein the first probe containsa detectable label.
 101. The method of claim 95, wherein the secondprobe contains a detectable label.
 102. The method of claim 86, whereinsaid cleaving produces a second fragment from the second probe whichdoes not associate with the second hybridization complex, and the methodfurther includes detecting said second fragment.
 103. The method ofclaim 102, wherein the second probe contains both (i) a fluorescent dyeand (ii) a quencher dye which is capable of quenching fluorescenceemission from the fluorescent dye when the fluorescent dye is subjectedto fluorescence excitation energy, and said cleaving severs a covalentlinkage between the fluorescent dye and the quencher dye in the secondprobe, thereby increasing an observable fluorescence signal from thefluorescent dye.
 104. The method of claim 102, wherein said secondfragment comprises one or more contiguous nucleotides which aresubstantially non-complementary to the target strand region.
 105. Themethod of claim 104, wherein said one or more contiguous nucleotidescomprise 1 to 20 nucleotides.
 106. The method of claim 102, whichfurther includes immobilizing the second fragment on a solid support.107. The method of claim 102, which further includes subjecting thesecond fragment to electrophoresis.
 108. The method of claim 102, whichfurther includes detecting the second fragment by mass spectrometry.109. The method of claim 102, which comprises detecting the secondfragment after the last cycle.
 110. The method of claim 102, whichcomprises detecting the second fragment during or after a plurality ofcycles.
 111. The method of claim 110, which comprises detecting thesecond fragment during all of the cycles.
 112. The method of claim 86,which further includes detecting the first hybridization complex afterat least one cycle.
 113. The method of claim 86, which further includesdetecting the second hybridization complex after at least one cycle.114. The method of claim 86, which further includes detecting the firstligated strand after at least one cycle.
 115. The method of claim 114,wherein said detecting comprises an electrophoretic separation step.116. The method of claim 114, wherein the first ligated strand isdetected by mass spectrometry.
 117. The method of claim 114, wherein thefirst ligated strand contains a fluorescent label.
 118. The method ofclaim 114, wherein the first ligated strand is immobilized on a solidsupport.
 119. The method of claim 118, wherein after the last cycle, thefirst ligated strand is immobilized on the solid support and detected.120. The method of claim 118, wherein said reacting comprises providingthe first probe or second probe immobilized on the solid support, sothat the first ligated strand is immobilized on the solid support. 121.The method of claim 86, wherein the first probe pair comprises a firstprobe and a second probe in covalently linked form, such that the firstprobe is covalently linked by its 5′ end to the 3′ end of the secondprobe by a linking moiety.
 122. The method of claim 121, wherein thelinking moiety comprises a chain of polynucleotides that is notsubstantially complementary to the target polynucleotide.
 123. Themethod of claim 86, wherein said reacting further comprises providing athird polynucleotide probe which is complementary to a sequence variantof a region to which either the first probe or second probe iscomplementary.
 124. The method of claim 123, wherein the thirdpolynucleotide probe and the first polynucleotide probe arecomplementary to alternative polymorphic sequences in the targetpolynucleotide strand region.
 125. The method of claim 124, wherein thetarget complementary sequences of the third polynucleotide probe and thefirst polynucleotide probe contain different 3′ terminal nucleotidesthat are complementary to alternative target nucleotide bases in thealternative polymorphic sequences.
 126. The method of claim 124, whereinthe first polynucleotide probe contains a first detectable label, andthe third polynucleotide probe contains a second detectable label thatis distinguishable from the first detectable label.
 127. The method ofclaim 126, wherein said labels are fluorescent labels.
 128. The methodof claim 123, wherein the third polynucleotide probe and the secondpolynucleotide probe are complementary to alternative polymorphicsequences in the target polynucleotide strand region.
 129. The method ofclaim 128, wherein the target complementary sequences of the thirdpolynucleotide probe and of the second polynucleotide probe containdifferent 5′ terminal nucleotides that are complementary to alternativetarget nucleotide bases in the alternative polymorphic sequences. 130.The method of claim 128, wherein the second polynucleotide probecontains a first detectable label, and the third polynucleotide probecontains a second detectable label that is distinguishable from thefirst detectable label.
 131. The method of claim 130, wherein saidlabels are fluorescent labels.
 132. The method of claim 86, wherein the5′ terminal base of said first region of the target strand region abutsthe 5′ terminal base of said first region of the target-complementarystrand region.
 133. The method of claim 86, wherein the 5′ terminal baseof said first region of the target strand region is separated from the5′ terminal base of said first region of the target-complementary strandregion by at least one nucleotide base.
 134. The method of claim 86,wherein said reacting further includes providing a second probe pairwhich is complementary to a second target polynucleotide strand region,the second probe pair comprising (i) a third polynucleotide probecontaining a sequence that is complementary to a first target region inthe second target strand region and (ii) a fourth polynucleotide probecomprising a sequence that is complementary to a second target region inthe second target strand region, wherein the second region is located 5′to the first region and overlaps the first region by at least onenucleotide base, under conditions effective for the third and fourthprobes to hybridize to the first and second regions in the second targetstrand region, respectively, forming a third hybridization complex ifthe second said target-complementary strand region is present in thesample.
 135. The method of claim 86, wherein the method furthercomprises reacting a sample with a plurality of different probe pairswhich are each designed to detect a different target polynucleotidesequence which may be present in the sample.
 136. The method of claim135, wherein said detecting comprises detecting at least one ligatedstrand produced by each different probe pair when the correspondingtarget sequence is present.
 137. The method of claim 136, whereinligated strands from different probe pairs are detected by massspectrometry.
 138. The method of claim 136, wherein ligated strands fromdifferent probe pairs are detected by electrophoresis.
 139. The methodof claim 138, wherein ligated strands from different probe pairs havedifferent electrophoretic mobilities.
 140. The method of claim 139,wherein ligated strands from different probe pairs contain detectablelabels which may be the same or different.
 141. The method of claim 140,wherein the labels are fluorescent labels.
 142. The method of claim 138,wherein ligated strands from at least two different probe pairs containdifferent fluorescent labels.
 143. The method of claim 135, whereinprior to step (a), the first probe or the second probe from one or moredifferent probe pairs is immobilized on a distinct solid support region.144. The method of claim 135, wherein the first probe or the secondprobe in each probe pair contains a distinct polynucleotide tag thatidentifies that probe pair.
 145. The method of claim 144, whichcomprises hybridizing species containing said tags to a plurality ofcorresponding tag complements which are immobilized on distinct solidsupport regions.
 146. The method of claim 145, wherein each distinctpolynucleotide tag is attached to the 5′ end of the first probe in eachdifferent probe pair.
 147. The method of claim 145, wherein eachdistinct polynucleotide tag is attached to the 3′ end of the secondprobe in each different probe pair.
 148. The method of claim 145,wherein said distinct solid support regions are located on asubstantially planar surface.
 149. The method of claim 145, wherein saiddistinct solid support regions are located on different beads.
 150. Themethod of claim 135, wherein for each probe pair, said cleaving producesa second fragment from the second probe of the probe pair which does notassociate with the third hybridization complex, and the method furtherincludes detecting the second fragment.
 151. The method of claim 150,wherein second fragments from different probe pairs are detected by massspectrometry.
 152. The method of claim 150, wherein second fragmentsfrom different probe pairs are detected by electrophoresis.
 153. Themethod of claim 152, wherein second fragments from different probe pairshave different electrophoretic mobilities.
 154. The method of claim 152,wherein second fragments from different probe pairs contain detectablelabels which may be the same or different.
 155. The method of claim 154,wherein the labels are fluorescent labels.
 156. The method of claim 150,wherein second fragments from at least two different probe pairs containdifferent fluorescent labels.
 157. The method of claim 150, wherein thesecond fragment from each different probe pair contains a distinctpolynucleotide tag that identifies the probe pair.
 158. The method ofclaim 157, which comprises hybridizing second fragments containing saidtags to a plurality of corresponding tag complements which areimmobilized on distinct solid support regions.